Cloning and sequencing of Fasciola hepatica cathepsin l2 DNA

Fasciolosis is a common parasite disease, caused by Fasciola hepatica thatbeing found throughout all regions of the world including Turkey, affecting bothhuman and animals. Sporadic cases of Fasciolosis have seen in Elazığ and itsseroprevalance in healthy individuals is reported to be 2.78%.Definitive diagnosis of fasciolosis is made by detection of eggs in the hostfaeces. But, detection of eggs is impossible in early infections, ectopic infections andfalse parasitosis, and serologic tests shouls be used to diagnose these conditions.Proteases represents the major parts of the excretory-secretory antigens of theFasciola hepatica and due to their specific antigenic nature they are potential candidatemolecules to be used for diagnosis and immunization methods. The aim of this studywas to clone and determine the sequence of cathepsin L2 gene.In the present study, we composed cathepsin L2 cDNA from RNA that obtainedF. hepatica from liver of naturally infected cattle by reverse transcription polymerasechain reaction (RT-PCR). After PCR amplification, cathepsin L2 cDNA was purifiedand was cloned into the pGEM-T Easy vector. The presence of cathepsin L2 gene wasconfirmed by PCR screening. This recombinant vector that named pGEM-T CL2 wastranfected into JM109 E. coli cells. So, pGEM-T CL2 was obtained from JM109 E. colicells by QIAGEN QIA Prep Kit and the presence of cathepsin L2 gene was confirmedby PCR screening. Cathepsin L2 cDNA was showed by agarose gel electrophoresis. Wedetermined DNA sequencing and performed phylogenetic analysisFor the first time, the cathepsin L2 of F. hepatica cloning was performed andsequencing from a Turkey isolate and nucleotide sequence showed 99% identity withpreviously published sequence of F. hepatica cathepsin L2 (GenBank accession no.U62289) by Dowd et al.Key words: F. hepatica, cathepsin L2, cloning, nucleotide sequencing,phylogenetic tree